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Duchatelet et al. Zoological Letters (2019) 5:9 https://doi.org/10.1186/s40851-019-0126-2

RESEARCHARTICLE Open Access Etmopteridae : dorsal pattern specificity and aposematic use Laurent Duchatelet1* , Nicolas Pinte1, Taketeru Tomita2,3, Keiichi Sato2 and Jérôme Mallefet1

Abstract Background: In the darkness of the ocean, an impressive number of taxa have evolved the capability to emit light. Many mesopelagic organisms emit a dim ventral glow that matches with the residual environmental light in order to themselves (counterillumination function). use their luminescence mainly for this purpose. Specific lateral marks have been observed in Etmopteridae sharks (one of the two known luminous families) suggesting an inter/intraspecific recognition. Conversely, dorsal luminescence patterns are rare within these deep- sea organisms. Results: Here we report evidence that spinax, Etmopterus molleri and Etmopterus splendidus have dorsal luminescence patterns. These dorsal patterns consist of specific lines of luminous organs, called photophores, on the rostrum, dorsal area and at periphery of the spine. This dorsal light seems to be in contrast with the counterilluminating role of ventral photophores. However, photophores surrounding the defensive dorsal spines show a precise pattern supporting an aposematism function for this bioluminescence. Using in situ imaging, morphological and histological analysis, we reconstructed the dorsal light emission pattern on these species, with an emphasis on the photogenic skin associated with the spine. Analyses of video footage validated, for the first time, the defensive function of the dorsal spines. Finally, we did not find evidence that Etmopteridae possess venomous spine-associated glands, present in and Heterondontidae, via MRI and CT scans. Conclusion: This work highlights for the first time a species-specific luminous dorsal pattern in three deep-sea lanternsharks. We suggest an aposematic use of luminescence to reveal the presence of the dorsal spines. Despite the absence of apparatus, the defensive use of spines is documented for the first time in situ by video recordings. Keywords: Etmopteridae, Dorsal pattern, Bioluminescence, Intraspecific recognition, Aposematism, Spines

Background organs, called photophores, mainly present on the ventral Deep in the ocean, a great many taxa have evolved the skin epidermis [7, 9–11]. The photophore structure, con- capability to emit light [1, 2]. This phenomenon, called served in the genus Etmopterus, is composed of a “deep” bioluminescence, is a mechanism whereby organisms emit pigmented sheet of embedded cells responsible for the visible light by biochemical reactions [1, 3, 4]. Functions of light emission, called photocytes, surmounted by an bioluminescence are mainly divided into three categories: iris-like structure topped externally by one or two lens cells , avoid predation (interspecific) and intraspecific [12–14]. Shark luminescence has been suggested to have communication [1, 3–6]. Among these organisms, sharks several ecological roles. Firstly, like a large number of are the first to utilize this phenomenon [1, 7, 8]. mesopelagic organisms emitting a continuous ventral glow Currently, there are two families of luminous deep-sea similar to the down-welling light, lanternsharks use their sharks (Etmopteridae and Dalatidae) which are capable of ventral light to disrupt their silhouette and avoid being emitting a blue-green light (from 460 to 486 nm according seen by predators swimming below; this is the counterillu- to the species) thanks to thousands of tiny luminous mination mechanism [1, 6, 10, 15]. Secondly, interspecific and intraspecific communication have been suggested: (i) * Correspondence: [email protected] the presence of species-specific lateral flank marks may 1 Laboratory, Earth and Life Institute, Catholic University of provide a way to facilitate reproductive isolation hence the Louvain, Place Croix du Sud 3, 1348 Louvain-la-Neuve, Belgium Etmopterus Full list of author information is available at the end of the article high species richness in genus [8, 16], while (ii)

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Duchatelet et al. Zoological Letters (2019) 5:9 Page 2 of 10

is observed in the Etmopteridae species facilitates the detection of moving objects in the inferior [12]. Aposematism is also suggested, provided by the spine visual field [34]. associated photophore luminescence [17]. Our results reveal the presence of a dim blue-green Previous studies have demonstrated that shark spines light from the dorsal epidermis for these sharks. We re- fulfill numerous functions, these include improving hydro- port a species-specific dorsal pattern of photophores that dynamics of the organism and serving as a mechanism for may be utilized for species recognition, schooling, and defense. The presence of venomous glands associated with other intraspecific communication. We also provide de- the posterior side of the spine in Heterodontidae and tails of the specific luminous pattern associated with the Squalidae are evidence for this function [18–22]. However, spine in different Etmopteridae species. In this study, the there is now in situ evidence for a defensive function of first evidence of dorsal spine in a defensive use, is the dorsal spine in Etmopteridae. In contrast to ventral recorded by in situ video footages of Etmopteridae luminescence, dorsal luminescence is rare in the ocean sharks. While CT and MRI scan images indicate that and has received less interest, probably because it is easily Etmopteridae sharks do not show the presence of a detectable in contrast to the darker background from the venomous gland associated with their spines. deeper waters underneath the organism. This dorsal pat- tern is usually utilized for predation [1, 5], as indicated by the dorsal lure located above the jaws in anglerfish species Materials and methods [23] or for an anti-predatory function, where the light acts Etmopteridae sampling as an aposematic warning signal [24–26]. The three elasmobranch species come from two different In this study, we investigated dorsal light emission in regions. E. spinax, is mainly found in the north-east part of three deep-sea shark species from the Etmopteridae family: the Atlantic, and were collected in the Raunefjord (60°15′ the velvet belly lanternshark, Etmopterus spinax (Linnaeus, 54″ N; 05°07′46″ E) next to Bergen in Norway during 1758); the slendertail lanternshark, Etmopterus molleri winter 2017. A total of 31 specimens were sampled during (Whitley, 1939); and the splendid shark, Etmopterus this field session. They were caught using a deep-long line splendidus (Yano, 1988). These three species are small at a depth ranging from 180 to 250 m. Specimens were deep-sea sharks (see Table 1) occurring at depths ranging transferred in a dark cold tank (4 °C) and brought to the from 200 to 500 m, where sunlight dimly penetrates the Espegrend marine field station where they are kept alive in [27–29]. Due to their small sizes, these sharks a seawater tank placed in a cold dark room (4 °C) until are preyed upon by larger organisms, such as other elasmo- manipulations. branch species including Dalatias licha, cookei E. molleri and E. splendidus were collected in the East and large species ([30–33], JM personal China Sea (26°28′94″ N; 127°41′20″ E) near the coast communication). Lanternsharks are characterized by miner- of Okinawa Island (Japan). They were fished using a alized spines in front of dorsal fins, a subterminal notch on bottom hook-and-line method at a depth ranging the dorsal part of the caudal fin and a specific pattern of light from 480 to 510 m. Data on E. splendidus and E. organs on the ventral and lateral body surface [11–13, 17]. A molleri were collected during the seasons win- recent study highlighted the specialization of the visual ter 2011 and winter 2016, respectively. Three speci- system in luminous sharks compared to mens of E. splendidus and 24 specimens of E. molleri non-luminous species: (i) longer rod outer segments, were collected. All specimens were transferred to higher rod densities and larger :body size ratio, these oxygen saturated plastic bags filled with seawater and are in favor of high light sensitivity; (ii) maximal absorp- transferred in a refrigerated box to the Okinawa tion wavelengths in visual (484–491 nm) for per- Churaumi Aquarium where they were kept alive in a ception of bioluminescent emissions (476 nm E. cold dark tank filled with seawater (13 °C) until splendidus,486nmE. spinax and 488 nm E. molleri)and manipulations. downwelling light; (iii) the presence of a secondary dorsal Data on the collected specimens are summarized in arch with a high density of rods in the retina which Table 1.

Table 1 Mean morphological values. N: number of specimens; ♀: female; ♂: male Species N Total length (cm) Fork length (cm) Pre-caudal length (cm) Weight (g) Etmopterus spinax 25 ♀ 46.3 ± 1.1 39.6 ± 1.1 35.6 ± 1.1 416.1 ± 23.8 6 ♂ 40.0 ± 1.3 35.2 ± 1.3 31.3 ± 1.3 248.1 ± 34.2 Etmopterus molleri 15 ♀ 42.7 ± 2.1 36.7 ± 1.9 33.9 ± 1.4 217.6 ± 43.1 9 ♂ 40.1 ± 0.9 33.8 ± 0.9 31.6 ± 0.7 165.6 ± 17.2 Etmopterus splendidus 3 ♂ 21.9 ± 1.6 19.6 ± 1.2 17.5 ± 0.9 / Duchatelet et al. Zoological Letters (2019) 5:9 Page 3 of 10

Dorsal luminescence pattern analysis spine [18–20, 22], magnetic resonance imaging (MRI) Dorsal photos of luminous and living shark were taken data of the E. spinax spine and the associated tissues with a Sony alpha 7S II (Sony Corporation, were obtained thanks to a Bruker Biospec 11,7 T (Bruker Japan), these images were analyzed, digital noise was BioSpin, Ettlingen, Germany) in order to visualized the removed using Photoshop software (Adobe; San Jose, presence/absence of a specific venomous structure. A CA, USA). Close-up images of the photogenic structure bird-cage coil with an internal diameter of 40 mm was associated with the dorsal spines was also completed used in emission/reception mode. The run sequence was with the same software. Flash type with the following parameters: TE: 3.2 ms; TR: 320 ms; FA: 25°; matrix size: 396 × 396; field of vision of Spine-associated luminous structure analysis 30 × 30 mm2; ten non-continuous slides separated from Since photophores located on the (SAP) 350 μm (center to center); resolution: 76 × 76 × 250 μm3; highlighting the spine were documented in E. spinax,we number of repetitions: 700. Computed tomography (CT) analyzed the structure and orientation of photophores data of E. spinax were acquired using a cone beam located around the spines in different Etmopteridae micro-CT scanner (NanoSPECT/CT, Bioscan inc., Wash- species with the aim to compare arrangements among ington D.C., USA) with the following characteristics: species. spatial resolution: 48 μm; X-ray tube voltage: 45 kVp; Captive sharks were euthanized by a knock on the number of projections: 360; exposure time: 1000 ms. The chondrocranium followed by an incision at the level of CT projections were reconstructed with a voxel size of the spinal cord. The local rules for experimental 0.111 × 0.111 × 0.11 mm3 by ray-tracing based filtered care and the European regulation for research back projection. handling were followed. Shark dorsal skin, spine and fin were dissected and directly stored in 4% paraformalde- Video footage hyde phosphate buffer saline (PBS) for 12 h at 4 °C, and During the field session in November 2016, video foot- stored in PBS until further use. A 1.5 cm diameter skin age of E. molleri was collected at one location in the patch around the spine was removed and separated from oriental China Sea (26°34′94″ N; 127°45′20″ E). Two the spine. For histological analyses, dorsal skin, fin epider- deployments occurred at depths of 500 and 540 m. Each mis and skin patches were bath in PBS with increasing su- video device was on the seabed during a period of 2 h. crose concentrations (10% for 1 h, 20% for 1 h and finally, The underwater video system was designed by ourselves. overnight in 30% sucrose), embedded in O.C.T. compound Video footage was taken by a GoPro Hero 4 (GoPro, (Tissue-Tek, The Netherlands) and finally, rapidly frozen Inc., San Mateo, CA, USA) placed in a special under- at − 80 °C. Thin sections (10 μm)werecutwithCM3050S. water case, benthic 2 (Group B Distribution Inc., Jensen Leica cryostat microtome (Germany) and were laid on Beach, FL, USA) and fixed on the metal frame. The bait Superfrost-coated slides (Thermo Scientific, Waltham, consisted of 1 kg of and in a metal MA, USA) and left overnight to dry. Slides were analyzed cage fixed to the frame by a steel bar. Lighting was pro- using an epifluorescence microscope and a light micro- vided by LED light in a housing, GPH-1750 M (Group B scope (Leitz Diaplan, Germany) equipped with a Nikon Distribution Inc.) and provided us a clear view until four Coolpix 950 camera (Nikon, Japan). General morphology meters and did not appear to disturb shark behaviors. The of the photophore, distance and the inclination angle (α) depth was recording using the sonar system of the boat. measured in relation to the spine location were all described. The photophore inclination angle (light path- Results way) was evaluated by taking the difference between the Dorsal luminescence pattern perpendicular to the iris opening as the reference axis and Analysis of different dorsal luminous pictures allowed us the line passing through the central point of the largest to build schematic drawings of the dorsal side of each lens. Photophore distance was measured from the center species (Fig. 1). Analyses of these patterns for each spe- of the light organ to the base of the spine. These two mea- cies revealed some luminous arrangements common to surements were taken on pictures via ImageJ software [35]. all species and others species-specific patterns: (i) the Statistical analyses were performed with JMP® software three luminous longitudinal lines from the back of the (JMP®, Version 13. SAS Institute Inc., Cary, NC, 1989– head to the beginning of the caudal fin were common 2007.). The Gaussian distribution respected an ANOVA for the three species studied (Fig. 1a-f), even if the me- followed by a Tukey-test to reveal significant differences. dian line of E. splendidus was denser than median line of the two other species; (ii) a relatively brighter luminous Computed tomography and MRI analyses mark located on the ventral side of the caudal fin dorsal Knowing that spine associated venom glands were de- lobe was observed; (iii) on the rostrum dorsal side, despite tected as soft tissue located at the posterior side of the many different patterns, the photophore aggregation Duchatelet et al. Zoological Letters (2019) 5:9 Page 4 of 10

Fig. 1 Dorsal luminescent patterns of E. spinax, E. splendidus and E. molleri. Schematic view of the dorsal patterns; images of dorsal luminous patterns and head close-up; schematic representation of the dorsal head luminous patterns of E. spinax (a, d, g, j), E. splendidus (b, e, h, k)andE. molleri (c, f, i, l) respectively. 1ds: first dorsal; 2ds: second dorsal; eb: eyeball; gl: gills; ns: nostril; pf: pectoral fin; pw: pineal window; sp.: spiracle. Scale bars 8 cm (a, b, c) and 2 cm for (j, k, l) around nostrils was a common feature (Fig. 1g-l); (iv) a distinguish any sexual differences during our observa- brighter luminous spot appeared next to the dorsal spines tions, although we observed that transferring the shark in these three species. from a captivity tank to an aquarium induces a transient In addition to these similarities, many different luminous increase of bioluminescence lasting around 10 min. arrangements occurred between these three species, mainly focused on the rostrum and pectoral fins. Indeed, Spine-associated luminous structure we saw that E. spinax and E. molleri show aggregation of Close-up observations of the dorsal fin body region were the luminous organs around the , next to spiracles and performed to analyze spine-associated luminous struc- at the edge of gill slits. A luminous impala horn shape pat- ture for the three studied species (Fig. 2). Dissected tern was observed on the E. spinax head (Fig. 1g, j) and a spines with the 1.5 cm skin-associated allowed us to particularly luminous shape arrangement was visible on E. characterize two different spine-associated photogenic molleri head (Fig. 1i, l). The pineal window surrounded by cluster patterns. Claes et al., (2013) describes the pat- a circle of luminous dots from which radiating lines terns in E. spinax, which consists of spine associated connecting spiracle, gills slits, and dorsal lines were visible. photophore, called SAP. These photophores are precisely Between this window and the nostril, a luminous V shape localized along the fin anterior ridge facing the spine and blotch were observed on the rostrum. Moreover, E. (Fig. 2a, d). This pattern was found and can be visualized molleri shows a specific luminous zone on pectoral fins through the thin dark strip on the antero-dorsal part of (Fig. 1c, f). the fin (Fig. 2a, d). At this location, the photophores are In contrast to these two species, E. splendidus oriented directly towards the spine and illuminate it possessed no specific luminous zones on the rostrum (Fig. 2g). E. molleri did not show any photophores on (Fig. 1h, k). the dorsal fin ridge (Fig. 2c, f, i) but careful examination The dorsal light emission is about one order of magni- of dorsal skin revealed the presence of luminous struc- tude dimmer than ventral light emission. We did not tures on the front and on each side of the spine base, Duchatelet et al. Zoological Letters (2019) 5:9 Page 5 of 10

Fig. 2 Spine-associated luminous structure analyses. Schematic view of the second dorsal spine; second dorsal spine images; second dorsal fin cross sections and skin base of the second spine of E. spinax (a, d, g, j), E. splendidus (b, e, h, k)andE. molleri (c, f, i, l) respectively. All sections are Masson’s trichrome stained. Cl: connective layer; D: dermal denticle; Ep: epidermis; L: lens; Ph: photocytes; Ps: pigmented sheath; SAP: spine associated photophores; SBAP: spine base associated photophores these photogenic structures were named spine base as- Spine structure and associated putative gland sociated photophore (SBAP) (Fig. 2c, l). These photo- To visualize a putative venom apparatus in Etmopteri- phores group together in small lines all around the spine dae, MRI analysis was performed on fixed specimens; with a rostro-caudal orientation (Fig. 2c, f). The size of image analyses do not show any evidence of a soft tissue SBAP clusters in front of the spine measured 775 μm± between the posterior side of the spine and the dorsal 322 and side clusters were estimated as 443 μm ± 543 fin (Fig. 5a; Additional file 1). CT scan analyses did not (Fig. 3a). Longitudinal and transversal sections across allow to highlight any canal/duct on the anterior side of SBAP allowed us to describe this new photophore type the spine that can be used for venom injection into a (Fig. 3b, e). SBAP is mainly composed of numerous potential predator (Fig. 5b; Additional file 2). photocytes forming an antero-posterior elongated tube shape, surrounded by pigmented cells and surmounted Video footage by at least five lens cells (Fig. 3a, b, e). These SBAPs are The video footage filmed in Okinawa allowed us to high- localized at a mean distance of 4.75 ± 0.74 mm from the light the defensive function of the dorsal spine in spine base (n = 210 SBAP observed). To find out if these Etmopteridae (Fig. 6). The video shows a sharpnose photophore lines were able to illuminate the spines, the sevengill shark, Heptanchrias perlo (Bonnaterre, 1788), inclination angles (α) between the SBAP (Fig.3d, e) and catching an Etmopterus splendidus (Fig. 6a), attempting ventral photophores (Fig. 3c, d) were measured. Ventral to bite it twice (Fig. 6b, c, d), the shark then opened its photophores showed an angle of 1.6° ± 0.8 (n = 42) jaws widely (Fig. 6e, f, g) letting the Etmopterus escape significantly different from right side SBAP presenting a (Fig. 6h, i). The original recording is provided as value of 29.1° ± 7.2 (n = 36) and left side, a value of 39.4° Additional file 3. ± 9.1 (n = 50) (Fig. 4; Tukey p < .001). Regarding E. splen- We also observed sequences where a sevengill shark didus, no specific spine associated photophores (SAP/ attempts to catch a lanternshark by the tail avoiding the SBAP) could be highlighted neither on the dorsal fin nor spine sting, as shown on the Additional file 4. During at the level of the spine base skin (Fig. 2b, e, h, k). field collections, caught lanternsharks were observed on Duchatelet et al. Zoological Letters (2019) 5:9 Page 6 of 10

Fig. 3 Spine base associated photophore (SBAP) close up structure. a External view of SBAP showing lens cells and the pigmented crown. b Transversal section of SBAP showing the internal structure of this new photophore type. c Longitudinal section of ventral photophore with a mean inclination angle <3°.d Schematic shark illustration showing ventral and SBAP inclination angles (e) Longitudinal section of SBAP with a mean inclination angle ±35° turned toward the spine. Red crosses correspond to the center of the biggest lens cell; ɑ: angle; Ep: epidermis; I Ch: isolated ; Cl: connective layer; L: lens; M: muscle; Pc: pigmented crown; Ph: photocytes; Ps: pigmented sheath; Sp: spine the hook with the tail cut off or the belly showing bite may have contributed to the large evolutionary radiation signs (Additional file 5). and speciation occurring in the Etmopterus genus [8, 16]. The dorsal lines already described in daylight by taxono- Discussion mists were never referred as bioluminescent lines [40–42]. Among luminous organisms of the , Thespecies-specificpatternscouldbeausefulfeaturefor dorsal luminescence is rare, as it seems counter-intuitive the Etmopteridae species determination/, and to produce a dorsal luminous signal making the emitter therefore used as a new morphological phylogeny criterion. highly visible against the darkness of the deeper waters, However, Claes et al. (2013) have suggested the dorsal while most organisms produce a ventral light to counter- aposematic function of light for E. spinax, where the illuminate and escape from the sight of predators [6, light from SAP (spine associated photophore) is strongly 36–38]. We observed specific rostrum and dorsal light transmitted by the dorsal spine making it visible for po- patterns, which may be utilized for intraspecific commu- tential predators. Our results show that handling the nication (schooling, mating), similarly to the dorsal cau- lanternshark species induces an increase of biolumines- dal gland of certain Myctophidae [39]. Assuming cence and the presence of a brighter spot of lumines- this intraspecific function, we suggest that this dorsal lumi- cence surrounding the spine areas, these findings agree nous pattern, like the specific flank marks of Etmopteridae, with this aposematic function. However, in E. molleri we Duchatelet et al. Zoological Letters (2019) 5:9 Page 7 of 10

Fig. 4 Mean inclination angle between iris perpendicular and the lens cells axis of left and right spine base associated photophores (SBAPs) and ventral photophores found a new elongated photophore type with numerous species [44]. The use of conspicuous signals to warn lenses whose orientation points towards the dorsal spine, predators of unprofitability, aposematism [45–48], has we call these spine base associated photophore or SBAP. been suggested for bioluminescent organisms in terres- These are much larger than the photophore commonly trial and oceanic environments [17, 24–26, 49–51]. described for Etmopteridae [7, 14, 43], and may repre- The presence of a venomous gland at the spine base in sent a cluster of numerous photophores aiming to light two shark families (Squalidae and Heterodontidae) was up the spine. Consistent with the phyl- considered proof of a defensive function of this spine ogeny, our results reveal a morphologically divergent [18–20, 22]. Despite that no evidence of such gland was of photophore (SAP/SBAP) within Etmopteri- shown by MRI and CT scan analysis at the level of the dae in order to reach a convergent functionality, dorsal spines in Etmopteridae, our video recordings and aposematism. The primary homology hypothesis seems images are the first in situ validation of a defensive use unlikely due to the positioning of the three studied of the dorsal spines. Attacks by predators at the level of

Fig. 5 Spine structure. Spine structure analyses by MRI image showing any evidence of soft tissue at the posterior side of the spine (red arrowhead) (a). Internal spine structure analyses by micro CT scan showing any evidence of a duct/canal at the posterior side of the spine hard tissue (calcified structure), red arrowhead showing the calcified tissue at the posterior side of the spine without any duct/canal (b). In both analyses, the only hollow on the spine, at the center, is filled with cartilaginous matrix Duchatelet et al. Zoological Letters (2019) 5:9 Page 8 of 10

Fig. 6 Schematic time-lapse of hunting behavior of H. perlo on an E. splendidus where the spine defensive function is illustrated (images taken from video footage see supplemental materials) the belly and tail of Etmopteridae seem to indicate that Additional files learning behavior has led predators to specifically avoid dorsal spines during predation attempts. Additional file 1: Animated GIF of MRI transversal section of Etmopterus spinax at the level of spine base, going from the tip to the base of the spine. (GIF 557 kb) Conclusion Additional file 2: Animated GIF of CT scan sagittal section of This work highlights for the first time a species-specific Etmopterus spinax dorsal spine and fin, starting from the body (1) till the luminous dorsal pattern in three deep-sea lanternsharks. end of the spine (4) and backward. (GIF 74 kb) New photophore assemblages were described and their Additional file 3: Video recording of Heptanchrias perlo attack on an arrangement suggests an aposematic use of lumines- Etmopterus splendidus body. (MOV 6040 kb) Additional file 4: Video recording of Heptanchrias perlo attack on an cence to reveal the presence of the dorsal spines. In Etmopterus molleri tail. (MOV 2448 kb) Etmopteridae, a morphological divergence might be in- Additional file 5: Pictures of injured E. molleri collected by deep sea rod volved in a convergent function, aposematism. Despite the fishing (A) Tail cut; (B) ventral side open; (C) closer view of B; (D) another absence of venomous apparatus, the defensive use of spines occurrence of ventral bite. Scale bar A = 3 cm, B –C –D = 4 cm. (TIF 19094 kb) is documented for the first time by in situ video recordings. Development of highly sensitive underwater video record- Acknowledgements ing devices could allow footage of bioluminescence to be We would like to thank T. Sorlie from the Espegrend Marine Biological Station (University of Bergen, Norway) for the help during E. spinax collection, the recorded, revealing the use of living light by deep-sea sharks husbandry staff from the Okinawa Churaumi Aquarium for the help provided during encounters with conspecifics or predators. during E. molleri and E. splendidus field sampling. We acknowledge the Louvain Duchatelet et al. Zoological Letters (2019) 5:9 Page 9 of 10

Drug Research Institute and the Institute of Neuroscience for the help they 7. Claes JM, Nilsson DE, Straube N, Collin SP, Mallefet J. Iso-luminance provide in MRI and CT scan analyses, respectively. Finally, we thank Muriel counterillumination drove bioluminescent shark radiation. Sci Rep. 2014;4: Blondeau for the time-lapse drawing provided. The authors want to acknowledge 4328. https://doi.org/10.1038/srep04328. the anonymous reviewers whose comments improve the present manuscript. 8. Straube N, Iglésias SP, Sellos DY, Kriwet J, Schliewen UK. Molecular phylogeny and node time estimation of bioluminescent lantern sharks Funding (: Etmopteridae). Mol Phylogenet Evol. 2010;56(3):905–17. This work was supported by a grant from the Fonds de la Recherche Scientifique https://doi.org/10.1016/j.ympev.2010.04.042. (FRS-FNRS, Belgium) to L.D., N.P., JM. 9. Claes JM, Mallefet J. Early development of bioluminescence suggests camouflage by counter-illumination in the velvet belly lanternshark – Availability of data and materials Etmopterus spinax (Squaloidea: Etmopteridae). J Fish Biol. 2008;73(6):1337 Please contact author for data requests. 50. https://doi.org/10.1111/j.1095-8649.2008.02006.x. 10. Claes JM, Aksnes DL, Mallefet J. Phantom hunter of the fjords: camouflage by counterillumination in a shark (Etmopterus spinax). J Exp Mar Biol Ecol. Authors’ contributions 2010;388(1):28–32. https://doi.org/10.1016/j.jembe.2010.03.009. L.D., N. P., T.T. and J.M. collected data, L.D., N. P. and J.M. contributed to data 11. Ebert DA, Fowler SL, Compagno LJ. Sharks of the world: a fully illustrated analysis and article preparation, T.T. and K.S. provided access to the field and guide. Plymouth: Wild Press; 2013. revised the manuscript. All authors reviewed the final version and agree to 12. Claes JM, Mallefet J. Ontogeny of photophore pattern in the velvet belly final article submission. lanternshark, Etmopterus spinax. Zoology. 2009;112(6):433–41. https://doi. org/10.1016/j.zool.2009.02.003. ’ Authors information 13. Claes JM, Sato K, Mallefet J. Morphology and control of photogenic L.D. and N.P. are PhD students under a FRIA fellowship; T.T. is under Researcher structures in a rare dwarf pelagic lanternshark (Etmopterus splendidus). J Exp fellowship from Okinawa Churashima Foundation Research Center; K.S. is Deputy Mar Biol Ecol. 2011;406(1):1–5. https://doi.org/10.1016/j.jembe.2011.05.033. Director General of the Okinawa Churaumi Aquarium; and J.M. is Research 14. Renwart M, Delroisse J, Claes JM, Mallefet J. Ultrastructural organization of Associate to FRS-FNRS. This paper is a contribution to the Biodiversity Research lanternshark (Etmopterus spinax Linnaeus, 1758) photophores. Zoomorphology. 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